1. Field of the Invention
The present invention relates to new plasmids from Corynebacterium glutamicum which are compatible with one another and to plasmid vectors (shuttle vectors) derived therefrom.
2. Background Information
Plasmid vectors are an essential prerequisite for improving bacterial strains by means of genetic engineering. The construction of plasmid vectors for Corynebacterium and Brevibacterium is based, in general, on cryptic plasmids which can be found in this group of bacteria.
Plasmid vectors for Corynebacterium and Brevibacterium can be used to clone genes involved in biosynthesis of amino acids, to express the corresponding gene product or enzyme to an increased degree and to improve the excretion of amino acids therewith. For example, the separation of L-lysine by means of Corynebacterium glutamicum can be improved by the cloning and overexpression of the phosphoenol pyruvate carboxylase gene of Corynebacterium glutamicum (European patent application No. 89 114 632.6).
Plasmids occur only very infrequently in the group of coryneform bacteria which excrete amino acids, even if the opposite impression might arise upon surveying the literature. As it turns out, upon closer examination, many plasmids described under different names exhibit so many identical qualities that they are to be considered as identical.
Examples of such plasmids are pAM286 from C. glutamic AJ11560 (EP-A-0 77 548); pAM330 from B. lactofermentum ATCC13869 (EP-A-0 77 548); pBL1 from B. lactofermenum ATCC21798 [Santamaria, R. et al., J. Gen. Microbiol. 130, pp. 2237-2246 (1984)] and pX18 from B. lactofermentum ATCC21086 [Yeh, P. et al., Gene 47, pp. 301-308(1986)].
The same holds true for the plasmids pHM1519 from C. glutamicum ATCC13058 (EP-A-0 78 537), pCG1 from C. glutamicum ATCC31808 (EP-A-0 58 889), pRN 3.1 from C. glutamicum ATCC39269 (DE-A-3402876) and pSR1 [Yoshihama, M. et al., J. Bact. 162, pp. 591-597 (1985)] from C. glutamicum ATCC19223. The high coincidence of the data published about these particular plasmids also shows that identical plasmid species must be involved. Martin, J. F. et al., [Bio/Technology 5, pp. 137-146 (1987)] confirm this assumption.
However, the number of publications and patent applications on this topic shows that there is much interest in a multiplicity of plasmids which are suitable for developing cloning systems for coryneform bacteria.
Plasmids which can exist in a cell adjacent to one another are of particular significance for the development of strains which excrete, for example, amino acids. Such compatible plasmids permit the simultaneous introduction of combinations of biosynthetic genes in a microorganism. Using compatible plasmids the desired enzyme activities can be increased and, in this manner, the formation of the desired product such as, for example, L-threonine or L-lysine can be improved. In particular, it would be especially advantageous to use compatible plasmids one of which exhibits a high copy number and the other a low copy number, in order to achieve a balanced overexpression of the particular genes while avoiding any unnecessary strain on the host.
While compatibility is important, at the same time, the stability of the plasmids used must, of course, be sufficient. As is known from the literature, however, there are no compatibility studies (Martin, J. F. et al., loc. cit., p. 139) or any investigations regarding the stability of plasmid vectors.